The development of a vaccine against human cytomegalovirus (HCMV) is a major public health priority. Of particular urgency is the need to protect newborn infants from the devastating consequences of congenital HCMV infection, including mental retardation, cerebral palsy, and deafness. Recently, a subunit vaccine based on recombinant HCMV glycoprotein B (gB) showed efficacy in a clinical trial. However, it is not clear that a vaccine based on a single HCMV protein will provide robust, long-term protection, particularly for women in their childbearing years. A potential significant advantage of the live, attenuated approach to HCMV vaccines is that such vaccines could elicit an immune response that mimics natural immunity and hence provide broader protection to a wide variety of virus-encoded targets of the host immune response. However, such vaccines carry the theoretical risk of establishing latency, reactivating under conditions of immune suppression, or causing chronic HCMV-associated diseases. An approach to generate an immunogenic, yet safe live vaccine is the deletion of viral genes that subvert host defense mechanisms to infection. Using the guinea pig model of congenital CMV infection, we propose to test this approach by targeting viral genes that subvert two key host defenses: MHC I homolog genes (NK cell evasins) and protein kinase R (PKR) evasins. In the previous funding period, we demonstrated efficacy and safety of a recombinant GPCMV vaccine which had a genomic deletion of a block of MHC I homologs. We propose to identify the key viral gene involved in this attenuation and to test our hypothesis that this gene product evades host defenses by inhibition of the NK cell response (aim 1). We hypothesize this virus will demonstrate superiority immunogenicity over wild-type virus, as assessed by cytokine response, CD8+ response, and antibody titer. A recombinant virus deleted of the MHC I function, ?(MHC, will be evaluated for attenuation in animals, and immune mechanisms will be confirmed by immune assays and NK depletion studies. In the previous funding period we have also identified a GPCMV gene involved in the inactivation of PKR. We propose to characterize this and other PKR evasins (aim 2) toward the goal of generating a deletion virus incapable of inhibiting the PKR response ((PKR). We will combine PKR and MHC mutations in a single attenuated virus construct that we predict will yield a safe, highly attenuated vaccine with improved immunogenicity. Despite tight innate immune control, this (MHC/(PKR construct is hypothesized to elicit strong, long-lasting protective immunity. This concept will be further tested in the GPCMV congenital infection model by evaluating and comparing the (MHC and (MHC/(PKR vaccines (aim 3). We hypothesize these vaccines will demonstrate equal or superior protection to infection with wild-type (control) virus against congenital infection and disease. By identifying optimal attenuated vaccine strategies in the GPCMV model, the translational prospect of genetically designing a vaccine for HCMV that has strong immunogenicity and improved safety will emerge.